Highly controllable electroporation and applications thereof

ABSTRACT

The controllable electroporation system and method described herein allows control over the size, the number, the location, and the distribution of aqueous pores, thus increasing flexibility of use. The herein described system and method for controllable electroporation generally employs at least two actuating sub-systems and sub-processes. One sub-system and sub-process employs a relatively broad effect in order to weaken the membrane, a broad effect sub-system. Another sub-system and sub-process employs a relatively narrow effect in order to localize the position of the pore in the membrane, a narrow effect sub-system.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 60/439,387 filed on Jan. 10, 2003, which isherein incorporated by reference

BACKGROUND

The membrane of a cell serves the vital function of partitioning themolecular contents from its external environment. The membranes arelargely composed of amphiphilic lipids, which self-assemble into highlyinsulating structures and thus present a large energy barrier totrans-membrane ionic transport.

However, the lipid matrix can be disrupted by a strong external electricfield leading to an increase in trans-membrane conductivity anddiffusive permeability, a well-known phenomenon known aselectroporation. These effects are the result of formation of aqueouspores in the membrane. More particularly, electroporation processinvolve permeation of cell membranes upon application of short durationelectric field pulses, traditionally between relatively large plateelectrodes (Neumann, et al., Bioelectrochem Bioenerg 48, 3-16 (1999);Ho, et al., Crit Rev Biotechnol 16, 349-62 (1996)).

For example, FIGS. 2A and 2B show a conventional electroporation system20, whereby impression of an electric field (e.g., shown by closing acircuit 22) creates random pores 26 in a cell membrane 24. Thedistribution of such pores, in terms of size and number only, isdetermined by the strength and duration of the applied electric field.The stronger and the longer the electric field is applied, the morenumerous and larger the pores are. However, the exact location of suchpores cannot be controlled, and thus the final distribution of pores issomewhat random. Researchers lose control over where compounds areintroduced into the intracellular matrix, an oft-important ingredient inbiochemical pathways. Thus researchers often have to rely on the cell'sown natural mechanisms, a far slower and difficult process to utilize.

Electroporation is used for introducing macromolecules, including DNA,RNA, dyes, proteins and various chemical agents, into cells. Largeexternal electric fields induce high trans-membrane potentials leadingto the formation of pores (e.g., having diameters in the range of 20-120nm). During the application of the electric pulse, chargedmacromolecules, including DNA, are actively transported byelectrophoresis across the cell membrane through these pores (Neumann,et al., Biophys J 712 868-77 (1996)). Uncharged molecules may also enterthrough the pores by passive diffusion. Upon pulse termination, poresreseal over hundreds of milliseconds as measured by recovery of normalmembrane conductance values (Ho, 1996, supra).

This procedure is often used in laboratory settings to inject chemicaland biological compounds into a cell, avoiding the reliance on thecell's own protein receptors and trans-membrane channels for transportacross the cell membrane. This allows researchers to easily study thebiological affect of compounds, be it a potentially life-saving cancerdrug or a deadly biological toxin. However, current electroporationtechniques are limited.

Therefore, it would be desirable to provide a method and system toovercome these and other limitations of conventional electroporation.

SUMMARY

The above-discussed and other problems and deficiencies of the prior artare overcome or alleviated by the several methods and apparatus of thepresent invention for controllable electroporation. The controllableelectroporation system and method allows control over the size, thenumber, the location, and the distribution of aqueous pores, thusincreasing flexibility of use. The herein described system and methodfor controllable electroporation generally employs at least twoactuating sub-systems and sub-processes. One sub-system and sub-processemploys a relatively broad effect in order to weaken the membrane, abroad effect sub-system. Another sub-system and sub-process employs arelatively narrow effect in order to localize the position of the porein the membrane, a narrow effect sub-system.

The above-discussed and other features and advantages of the presentinvention will be appreciated and understood by those skilled in the artfrom the following detailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show operation of the controllable electroporationsystem described herein;

FIGS. 2A and 2B show operation of conventional electroporation systems;

FIGS. 3A-3D show a general embodiment of a cell injection system;

FIG. 4A-4B shows an example of a cell injection system;

FIG. 5A-5B shows an example of a cell injection system for an array ofcells;

FIG. 6 shows a separation device using multi-phospholipid layers andelectroporation;

FIG. 7 shows a separation device using multiple layers, electroporationand a microfluid array;

FIG. 8 shows a separation device using a single layer capapble of havingdifferent pores at different locations; and

FIG. 9 shows an exemplary electrode grid that may be used in variousembodiments described herein.

DETAILED DESCRIPTION

Herein described is an electroporation system and method providingcontrol over the size, the number, the location, and the distribution ofaqueous pores, thus increasing flexibility of use. Referring generallyto FIGS. 1A and 1B, the herein described system 10 and method forcontrollable electroporation of a membrane 12 generally employs at leasttwo actuating sub-systems and sub-processes. FIG. 1A shows a portion ofa membrane 12, and FIG. 1B shows the controllable electroporation system10 described herein.

Note that in general, one of the actuating sub-systems alone will notsuffice to open or create a pore 14 in the membrane—both actuatingsub-systems are employed, thereby functioning in a similar manner as alogical “and” gate. A broad effect sub-system 16 employs a relativelybroad effect in order to weaken the membrane, and the narrow effectsub-system 18 employs a relatively narrow effect in order to localizethe position of the pore 14 in the membrane 12. Employing both the broadeffect sub-system 16 and the narrow effect sub-system 18 enables highlylocalized and controlled electroporation and hence opening of pore 14.

Therefore, for example as compared to conventional electroporationprocesses of cellular membranes, described in the Background and withrespect to FIGS. 2A and 2B, in addition to the applied electric field ofconventional electroporation technology, the herein disclosedelectroporation system and method employs a narrow effect sub-system 18directed at a specific location on the cellular membrane, enablingpositional control over the pore 14. The narrow effect sub-system 18will excite the phospholipid molecules, thus reducing the amount ofenergy required from the broad effect sub-system 16 to create theaqueous pores. Thus, for example, a very weak electric field can beapplied to a system, in which typically, electroporation would notoccur. However, this weak electric field can open aqueous pores inplaces of the cellular membrane already excited by the laser beam.

The broad effect sub-system or sub-process 16 may be selected from anysuitable membrane weakening systems and/or processes. Such weakeningsystems and/or processes may be selected from the group consisting ofelectric fields (in certain preferred embodiments uniform electricfields), microwave energy, other electromagnetic radiation, relativelylow energy laser beams (i.e., lower energy than that required tocommence random electroporation), or any combination comprising at leastone of the foregoing weakening systems and/or processes. The energymagnitude of the broad effect sub-system or process 16 is generallylower than the energy magnitude of conventional electroporation systemswhereby random pore opening occur. Further, the area (e.g.,cross-sectional area) of the weakening systems and/or processes 16generally encompasses an area larger than the desired pore size. Incertain embodiments, this area encompasses the entire cell membrane oran array of cell membranes. In other embodiments, this area is a regionof a membrane.

The narrow effect sub-system or sub-process 18 may be selected from anysuitable membrane pore position localization systems and/or processes.Such position localization systems and/or processes may be selected fromthe group consisting of laser beams, electrode tips, or any combinationcomprising at least one of the foregoing position localization systemsand/or processes. The area (e.g., cross-sectional area) of the positionlocalization systems and/or processes 18 is generally narrow, e.g.,corresponding to the desired dimensions of the pore opening. Thus, forexample, controlled pore openings having of sub-micron or nanometer(e.g., 1-100 nm) magnitude are enabled, since existing and developinglaser and electrode tip technologies are capable of suchsub-micron-scale and nano-scale dimensions.

Applications

Cell Injection

The herein described controllable electroporation system and process maybe used to inject macromolecules, including DNA, RNA, dyes, proteins andvarious chemical agents, in a controlled manner. Without intending tolimit the applications of the present controllable electroporationsystem, FIGS. 3A-5B show various embodiments of cell pore openingsystems employing the controllable electroporation system.

Referring now to FIGS. 3A-3D, a system 30 is shown for controllableinjecting macromolecules into a cell. FIG. 3A depicts the system 30including a mechanism 32 for holding a cell 24. Mechanism 32 is asuitable microrobotic device including associated Microsystems as aregenerally known in the biotechnology arts. Such as device 32 preferablyis capable of holding individual cells or controlled groups of cells.Further, mechanism 32 may also be used to obtain biological, electrical,optical, or other data from the cell 24.

Referring now to FIG. 3B, the system 30 is shown including the mechanism32 holding the cell 24, and a controllable electroporation systemincluding the broad effect sub-system 16 and the narrow effectsub-system 18, whereby the narrow effect sub-system 18 is focused at alocation on the cell 24 to induce opening of a pore 34.

Referring now to FIG. 3C, a macromolecule 38 is introduced, for example,via a nano-nozzle or other suitable injection device 36. When the narroweffect sub-system 18 and/or the broad effect sub-system 16 is removed,the pore will close, resulting in cell 24′ having macromolecule 38therein.

FIGS. 4A and 4B show one embodiment of a controllable electroporationsystem 40 for introducing controllably opening a pore 34 in a cell 24,e.g., for introduction of macromolecules as described above. The system40 includes a broad effect sub-system in the form of an electric fieldproducing apparatus 42, 44, 46, and a laser beam 48 from a suitablesource (not shown). The electric field producing apparatus is in theform of an electrode plate 42 coupled to a switchable (via a switch 44)voltage source 46. As shown, the laser beam may be focused, and theelectric field applied to active the pore opening mechanism, akin to alogical “and” circuit as described above.

With the system and method described with respect to FIGS. 3A-3D and4A-4B, researchers could only expose a few cells in a tissue constructto a biological compound and observe how the signal is propagated to itsneighboring cells. Alternatively, researchers could study if asymmetriccells such as neurons and gastrointestinal mucosa cells reactdifferently to compounds injected at different places.

Referring now to FIGS. 5A and 5B, a system 50 is shown that operatessimilar to that of FIGS. 3A-3D or 4A-4B in conjunction with an array 52of cells 24. When the broad effect sub-system 16 and the narrow effectsub-system 18 are operated, pores 34 are formed in the cells 24. Suchpores may be used for selective introduction of macromolecules into thecells 24.

Separation Device Referring now to FIGS. 6-10, various embodiments offiltration/separation devices are provided using the controllableelectroporation system herein.

FIG. 6 depicted one embodiment of a system 60, e.g., a molecular sieve.System 60 includes plural membrane layers 62, e.g., phospholipidbilayers. Each membrane layer 62 may be subject to a narrow effectsub-system during application of the broad effect sub-system,alternatively the location of the pores 64 may be predetermined uponassembly and or manufacture, e.g., with suitable micro- or nano-defects,or may be identical whereby different voltage levels at each layerdetermined the pore size. Application of different voltages (e.g. V1, V2and V3) at each layer creates a filter gradient from large pores 64 tosmall pores 64, allowing passage of molecules 66 through suitablelayers.

Using a phospholipid bilayer, which is extremely cheap to manufacture,the same filter 60 can be used repeatedly and adapted for any sizerequirements using electroporation and carefully controlling theelectric field that is applied. Instead of depending on multiplefilters, a single filter could be used and configured for any situation.

Referring to FIG. 7, a system 70 is shown with a molecular sievefunctioning similar to that of FIG. 6 associated with a biochip array 72having channels 74 therein. Channels 74 may serve to collectmacromolecules and molecules at each level based on size. Further,channels 74 may incorporate or be associated with a gradient system,e.g., pressure, thermal, electrical, or other gradient to inducemacromolecules and molecules toward the array 72. Array 72 may be anysuitable microfluidic or nanofluidic device. For example, methods ofmanufacturing such devices are described in Reveo Inc. PCT ApplicationNo. PCT/US03/37304 filed Nov. 30, 2003 entitled “Three DimensionalDevice Assembly and Production Methods Thereof”, which is incorporatedby reference herein.

FIG. 8 shows another example dynamic filtration device, wherein an arrayof lasers provides positional control over the pore openings. FIG. 80shows a filtration system 80 including a membrane layer 82 associatedwith a broad effect energy sub-system 16 and a narrow effect sub-system18. For example, the narrow effect sub-system 18 may be generated with alaser array 88. Alternatively, instead of an array of lasers 88, a beamsteering device may be incorporated, allowing use of only one lasersource. When a laser is activated from the array associated with acertain position on the membrane 82, a pore 84 will open. The size ofthe pores may be controlled by predetermined membrane characteristics,area or magnitude of the narrow effect energy sub-system, or magnitudeof the broad effect energy sub-system.

Cells, proteins, enzymes, DNA molecules, RNA molecules, and othermacromolecules or molecules may be collected via an array of containers86, e.g., on a suitable microfluidic device. Thus, separation device 80may be made extremely compact and highly flexible for any purpose.

FIG. 9 shows an example of an electrode suitable for providing the broadeffect energy sub-system in various embodiments shown herein. Byproviding electrodes in a grid pattern, a suitable field generatingsystem may be provided to allow access for various purposes includingthe narrow effect sub-system, macromolecule introduction, filtration, orany other purpose.

In addition to filtering based on size, the aforementioned separationdevices may also separate on the basis of ionic charge, since theapplied voltage will drive only one type of ions across the membrane.

While preferred embodiments have been shown and described, variousmodifications and substitutions may be made thereto without departingfrom the spirit and scope of the invention. Accordingly, it is to beunderstood that the present invention has been described by way ofillustrations and not limitation.

1. A system for controllable electroporation of a membrane comprising: abroad energy sub-system operably coupled to the membrane and a narrowenergy sub-system operably coupled to the membrane, wherein a pore isopened or created at a position corresponding to the position of thenarrow energy.
 2. The system as in claim 1, wherein the broad energysub-system is selected from the group of weakening systems consisting ofelectric fields, microwave energy, other electromagnetic radiation, lowenergy laser beams, or any combination comprising at least one of theforegoing weakening systems.
 3. The system as in claim 1, wherein theenergy magnitude of the broad energy sub-system is lower than the energymagnitude of electroporation systems without the narrow effectsub-system whereby random pore opening occur.
 4. The system as in claim1, wherein the area of the broad energy sub-system encompasses an arealarger than the desired pore size.
 5. The system as in claim 1, whereinthe area of the broad energy sub-system encompasses the membrane of acell.
 6. The system as in claim 1, wherein the area of the broad energysub-system encompasses membranes of an array of cells.
 7. The system asin claim 1, wherein the area of the broad energy sub-system encompassesa region of a membrane.
 8. The system as in claim 1, wherein the narrowenergy sub-system is selected from the group of position localizationsystems consisting of laser beams, electrode tips, or any combinationcomprising at least one of the foregoing position localization systems.9. The system as in claim 1, wherein the area of the narrow energysub-system corresponds to the dimensions of the pore opening.
 10. Thesystem as in claim 1, wherein the pore has sub-micron dimensions. 11.The system as in claim 1, wherein the pore has dimensions of about 100nanometers or less.
 12. A controllable electroporation systemcomprising: a broad energy sub-system operably coupled for providingbroad energy to the membrane and a narrow energy sub-system operablycoupled to the membrane, wherein a pore is opened when both the broadenergy sub-system and the narrow energy sub-system are activated. 13-23.(canceled)
 24. A cell pore opening system comprising: a microroboticdevice for holding a cell and a system as in claim 1 for controllablyopening a pore in the cell.
 25. The cell pore opening system as in claim24, wherein the broad energy sub-system comprises an electrode plate anda switchable voltage source.
 26. The cell pore opening system as inclaim 24, wherein the narrow energy sub-system comprises a laser.
 27. Acell pore macromolecule system comprising: a microrobotic device forholding a cell; a system as in claim 1 for controllably opening a porein the cell; and a macromolecule injection device for injecting amacromolecule into the cell via the pore. 28-29. (canceled)
 30. A systemfor filtering molecules or macromolecules comprising: a plurality ofmembrane layers, each membrane layer including a system as in claim 1for controllably opening a pore in the cell, each membrane layer openedto a different size to create a pore size gradient.
 31. A system forfiltering molecules or macromolecules comprising: a plurality ofmembrane layers, a broad energy sub-system associated with each membranelayer, wherein each layer includes a position having a defect wherebysaid position is closed without activation of the broad energysub-system and said position is opened upon activation of the broadenergy sub-system.
 32. The system as in claim 31, wherein said defect ateach layer controls the size of the pore.
 33. The system as in claim 31,wherein the magnitude of the energy of the broad energy sub-systemcontrols the size of the pore.
 34. A system for filtering molecules ormacromolecules comprising: a membrane layer including a system as inclaim 1 for controllably opening a pore in the cell, wherein the narrowenergy sub-system comprises an array of narrow energy sub-sub-systems.35. A system for filtering molecules or macromolecules comprising: amembrane layer including a system as in claim 1 for controllably openinga pore in the cell, wherein the narrow energy sub-system comprises anarray of lasers.
 36. A system for filtering molecules or macromoleculescomprising: a membrane layer including a system as in claim 1 forcontrollably opening a pore in the cell, wherein the narrow energysub-system comprises a beam steering device associated with a source ofelectromagnetic energy. 37-47. (canceled)